Background & objectives: An increase in Zika virus (ZIKV) epidemic during the last decade has become a major global concern as the virus affects both newborns and adult humans. Earlier studies have shown the impact of ZIKV infection in developing human foetus. However, effective in vitro model of target cells for studying the ZIKV infection in adult human neurons is not available. This study aimed to establish the use of human neuroblastoma cell line (SH-SY5Y) for studying an infection of ZIKV in vitro.Methods: ZIKV growth kinetics, viral toxicity, and SH-SY5Y cell vialibity were determined after ZIKV infection in SH-SY5Y cells in vitro. ZIKV-infected SH-SY5Y cells were morphologically analysed and compared with nonhuman primate Vero cells. Furthermore, the susceptibility of SH-SY5Y cells to ZIKV infection was also determined. Results: The results showed that ZIKV efficiently infects SH-SY5Y cell lines in vitro. Gradual changes of several cellular homeostasis parameters including cell viability, cytotoxicity, and cell morphology were observed in ZIKV-infected SH-SY5Y cells when compared to mock-treated or non-human primate cells. Interestingly, ZIKV particles were detected in the nucleoplasmic compartment of the infected SH-SY5Y cells.Interpretation & conclusion: The results suggest that ZIKV particle can be detected in the nucleoplasmic compartment of the infected SH-SY5Y cells beside the known viral replicating cytoplasmic area. Hence, SH-SY5Y cells can be used as an in vitro adult human neuronal cell-based model, for further elucidating the ZIKV biology, and highlight other possible significance of Zika virus distribution through nuclear localization, which may correlate to the neuropathological defects in ZIKV-infected adult humans.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that was first isolated from the serum of a Rhesus monkey in Uganda in 1947[1]. Zika virus is closely related to other members of Flaviviridae family including dengue virus (DENV), Yellow Fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV)[2]. The ZIKV is primarily transmitted through the bite of an infected Aedes mosquitoes in a sylvatic transmission cycle with non-human primates. Moreover, human-mosquito-human transmission cycle has been shown to increase the epidemic in urban and suburban areas involving two major Aedes species (Ae. aegypti and Ae. albopictus)[3]. Studies based on serosurveys have shown that distribution of ZIKV is not limited to Uganda, rather it has been reported from other countries including Egypt, India, Thailand, Vietnam, Philippines, Malaysia, and America[4].

In the past, ZIKV epidemics were low and severity ranged from mild to moderate in most the infected individuals which included malaise, headache, fever, maculopapular rash, and conjunctivitis[5]. Over the last decade, ZIKV has emerged from its relative obscurity to significant outbreaks as in Brazil[6]. Several reported cases have shown the association of ZIKV infection to featal microcephaly in newborns occurring at the first two trimesters of pregnancy[5],[7],[8]. Some reported cases in French Polynesia exhibited its association with serious neurological complications in adults such as Guillain-Barré syndrome, representing as a symmetric ascending flaccid paralysis[9]. Due to these serious complications and increase in outbreaks, WHO declared it as a ‘Public Health Emergency of International Concern’ on February 1, 2016[10].

Zika virus can be injected into human by ZIKV-in-fected female Aedes mosquito during blood feeding. Zika virus further infects the permissive cells of human skin. A previous study has shown that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are susceptible to ZIKV infection[11]. The study suggests that during the host cell infection, ZIKV use components of autophagy pathway to enhance their replication. Although, ZIKV infects human skin cells for their multiplication, little is known about how they cause human neuronal defects. Earlier in vitro and in vivo studies have shown that it infects human neuronal progenitor cells, upregulate innate immune receptor–Toll-like receptor 3 (TLR3); promote cell-cycle arrest, and apoptosis of neuronal progenitor cells. These studies suggest the possible role of neuronal progenitor depletion in neuropathology associated with ZIKV infection. They also showed that human neuronal cells are the direct targets for ZIKV, providing a direct link between ZIKV infection and neurological complications in human[12],[13],[14].

A recent study on ZIKV structure using cryoelectron microscopy (cryo-EM) has revealed that ZIKV exhibits structure similar to other viruses in a Flaviviridae family[15],[16],[17]. Viruses in a Flaviviridae family tend to enter the host cells via endocytosis, wherein the new virus progeny assembles in the endoplasmic reticulum (ER) of the hosts[18],[19]. Moreover, proteasomal degradation of type I interferon (IFN)-regulated transcriptional activator STAT2 has been shown to be induced upon ZIKV infection in various types of human cells (embryonic kidney and fibroblast cells) in vitro, similar to other flaviviruses[20]. The study suggests that inhibition of IFN signaling in ZIKV infected host cells could cause an impairment of host antiviral innate immunity and might be responsible for rapid viral emergence and pathogenesis of ZIKV in humans.

Despite the fact that ZIKV causes neuronal defects both in developing and in adult humans, most studies have focused on the impact of its infection to developing human brain, with less attention on adult neurons. Thus, there is still an urgent need of direct evidence from human adult neuronal cell-based model showing its negative impact on neuronal cell homeostasis. In the present study, a new strategic approach was used to investigate the infection route of ZIKV on adult human neuronal cells by using human neuroblastoma cell line (SH-SY5Y) in vitro and its impact on the growth of SH-SY5Y cells. The results indicated that ZIKV infection increases the replication rate of SH-SY5Y cells and attenuates their growth. Interestingly, the electron microscopy, showed that ZIKV is localized in the nuclear region in the infected SH-SY5Y cells apart from its natural behaviour of viral replication in the cytosol.

Material & Methods

Cell cultures

The human SH-SY5Y cell line, Rhesus macaca cell line (LLC-MK2), and African green monkey kidney (Vero) cells were cultured in a Dulbecco’s modified eagle medium (DMEM) supplemented with penicillin (20 units/ml), streptomycin (20 mg/ml), and 10% (v/v) heat-inactivated fetal bovine serum (all from Gibco, Gaithersburg, MD). The cells were maintained in a incubator with 5% CO2 at 37°C. Flasks with approximately 90% of confluence were used for subsequent analysis.

Zika virus growth kinetics on SH-SY5Y cells

Zika virus isolates (SV0127/14 and SV0010/15) were kindly provided by the Armed Forces Research Intitute of Medical Sciences (AFRIMS) and Ministry of Public Health, Thailand. The ZIKV was propagated in LLC-MK2 cells in a 6-well plate (5×105 cells/well) and cultured in DMEM under 5% CO2-enriched conditions. The virus titres expressed as plaque-forming units (PFU) were determined by plaque assay[21]. Briefly, after host cells were being adsorpted with a serially diluted virus solution for 1 h, the solution was replaced with fresh DMEM containing 2% FBS and 0.5% methyl cellulose (Sigma-Aldrich, St. Louis, MO). After five days post-infection, the medium was removed. The cells were fixed and stained with crystal violet solution containing of 1% crystal violet, 0.64% NaCl, and 2% formalin. Diluted viral solution at multiplicity of infection (MOI)-1 was chosen to further infect the SH-SY5Y cells.

SH-SY5Y cells were resuspended at a concentration of 5×105 cells/ml in appropriate medium with ZIKV at MOI of 1 and incubated for 1 h at 37°C. Then, the cells were washed once and resuspended with DMEM medium for further incubation. The cell-free supernatants were used to determine the virus growth kinetic by using conventional RT-PCR assay (viral identification) and viral plaque assay (viral titre) at different time intervals (3 h, 1, 2, 3, 4, and 5 days). Conventional RT-PCR was performed using Zika specific genome primers: Forward, 5'-TTGGTCATGATACTGCTGATTGC-3'; Reverse: 5'-CCTTCCACAAAGTC-CCTATTGC-3’. The expected PCR products size was determined at 77–100 bp.

SH-SY5Y cells viability assay after Zika virus infection

To assess SH-SY5Y cell viability, the number of viable cells after 3 h, 1, 2, 3, 4, and 5 days post-infection was determined by using trypan blue dye exclusion method[22]. The assay is based on the ability of intact viable cells to exclude trypan blue dye. Briefly, ZIKV-infected SH-SY5Y cells and mock-infected cells at 2×105 cells/well were cultured in 6-well plates. At pre-determined time points after infection, the cells were washed once with PBS and trypsinized to collect cell pellets by centrifugation at 300 g for 5 min, followed by re-suspending the pellets in PBS. Subsequently, 0.2 ml of the suspension was mixed with an equal volume of 0.4% trypan blue (Sigma-Aldrich, St. Louis, MO), incubated for 5 min and observed under a microscope by counting both total cell and stained cell numbers with haemocytometer.

SH-SY5Y cells cytotoxicity assay after Zika virus infection

Each group of SH-SY5Y cells (ZIKV-infected and mock) was seeded at 2×105 cells/well in a 96-well plate and cultured in DMEM supplemented with 10% FBS at 37°C with 5% CO2, until 85% confluency. At different time post-infection (3 h, 1, 2, 3, 4, and 5 days), MTT reagent (5 mg/ml; Sigma-Aldrich Chemie GmbH, Germany) was added to the cell medium and incubated at 37°C for an additional 4 h. The reaction was terminated by adding 150 μl dimethylsulfoxide/well (Sigma-Aldrich Chemie GmbH, Germany) and the cells were lysed for 15 min, with the plates gently agitated every 5 min. The absorbance values were determined using an enzyme-linked immunosorbent assay reader (Model 680; Bio-Rad, Hercules, CA) at 490 nm.

Morphological changes of ZIKV induced cytopathic effects (CPE) in SH-SY5Y cells were observed and compared with Vero cells at 3 h, 1, 2, and 3 days post-infection. The original photomicrographs were taken at 200x using a phase-contrast microscope. The CPE manifested by multinucleated giant cells, cell shrinkage, and foci of cell destruction in SH-SY5Y and Vero cultures following ZIKV infection at various time points were evaluated and scored (as described by Moore et al[23]): 0 (no CPE); +/-(enlargement of some cells in monolayer); 1+ (1–25% CPE); 2+ (25–50% CPE); 3+ (50–75% CPE); and 4+ (75–100% CPE).

After 12 h post-infection, the positive ZIKV-infected SH-SY5Y cells (as determined by conventional RT-PCR) were used to study the intracellular structure by electron microscopy. ZIKV-infected SH-SY5Y cells were primary fixed with 2.5% glutaraldehyde in 0.1 M sucrose phosphate buffer (SPB), pH 7.4 for 1 h. Cells were then washed with SPB for three times and fixed again with 1% osmium tetroxide in SPB for 1 h. The cells were again washed, dehydrated with grading ethyl alcohol, pre-in-filtrated with LR White® (Electron Microscope Sciences Inc., Hatfield) in 70% ethyl alcohol (2:1) and 100% LR White® for 1 h each at room temperature, and thereafter incubated in 100% LR White® for overnight at 4°C. The cells were embedded in capsule beams for 48 h polymerization at 65°C. After that, cells were sectioned at 80–90 nm using ultra-microtome (Leica). Finally, all sections were mounted on 200 mesh copper grids, stained with uranyl acetate and lead citrate. Images were observed and acquired using a HT7700 Toshiba’s electron microscope.

Statistical analysis

All experiments were done at least in triplicates. Data were represented as the means ± SE. Statistical significances between groups were assessed using a two-tailed Student t-test.

Results

SH-SY5Y cells are permissive for Zika virus infection and replication

To study the impact of Zika virus infection on human adult neurons, the virus propagated in LLC-MK2 cell lines (rhesus macaca) were passaged in human SH-SY5Y cell line (for determining the virus kinetics and host-cell growth). Although, the titre of ZIKV in the infected human is currently unidentified, our previously published study showed that ZIKV can infect primary human fibroblasts at MOI of 1[11]. Hence, ZIKV infections were preformed at MOI of 1 in SH-SY5Y cells, and the medium containing virus inoculum was removed after 1 h postinoculation period. The ability of these cells to produce viral progeny in vitro was evaluated by determining the virus titres in the supernatants of ZIKV-infected SH-SY5Y cells, using a standard plaque assay. The results showed a gradual increase in the production of virus particles in a post infection time-dependent manner. There was significant increase in virus particles after Day 1, post-infection (p<0.05). These results suggested that active viral replication occurred in the infected cells [Figure 1]a. The intracellular viral RNA was also quantified using conventional RT-PCR at different time intervals, post-infection. The results showed the presence of ZIKV RNA in the ZIKV-infected SH-SY5Y cells; however, it was not detected in mock-infected control as shown in [Figure 1]b. The viral RNA was detected at 3 h post-infection, with highest intensity between Day 1 and 3 correlated to the increasing viral titres. Taken together, these results indicate that SH-SY5Y cells are the target cells of Zika virus.

The impact of ZIKV infection on SH-SY5Y homeostasis was further evaluated by determining the cell number and percentage of cellular cytotoxicity. The amount of SH-SY5Y cells after infection with ZIKV at MOI of 1 or mock control at different intervals, was determined by trypan blue exclusion method. The results showed that the number of SH-SY5Y cells significantly decreased after infection with ZIKV in a time dependent manner compared to mock infection control [Figure 2]a. The evaluation results for the potential impact of ZIKV infection induced cellular cytotoxicity on SH-SY5Y cells indicated that the reduction of cell number is correlated to the increase in percentage of cytotoxicity after infection under the same experimental condition as determined by MTT assay [Figure 2]b. Therefore, ZIKV infected SH-SY5Y cells caused growth inhibition in this cell population by increasing the cellular cytotoxicity.

Human cells (SH-SY5Y) are more permissive for Zika virus than other primate cells (Vero)

To investigate the direct impact of ZIKV on human neuronal cell morphology, the photomicrographs of ZIKV-infected SH-SY5Y cells at different time interval post-infection was analysed and compared with ZIKV-infected Vero cells. It was observed that ZIKV induced morphological changes through CPE grading method in both SH-SY5Y and Vero cells in a time dependent manner [Figure 3]; however, no such changes were seen in mock controls of these cell types. The changes were more prominent in SH-SY5Y cells (CPE = 1+) than in the Vero cells (CPE = +/−) after Day 1 post-infection in the form of enlargement of cell size. The CPE induction was >75% in SH-SY5Y cells (CPE = 4+) while it was <50% in Vero cells (CPE = 2+) after Day 3 post-infection. The rapid morphological changes in SH-SY5Y cells after infection with ZIKV is correlated to a decrease in cell viability and an increase in cellular cytotoxicity [Figure 2]. These results suggest that ZIKV is highly permissive to SH-SY5Y cells rather than non-human (Vero) cells.

Zika virus is located both in both cytoplasmic and nucleoplasmic compartments of infected SH-SY5Y cells

The infection route of ZIKV was analyzed in ZIKV-infected SH-SY5Y cells by using electron microscopy. The electron micrograph revealed that ZIKV particles were present within the cytoplasmic compartments of SH-SY5Y cells 12 h post-infection [Figure 4]b and [Figure 4]c. Interestingly and surprisingly, ZIKV particles were also observed in the nucleoplasmic compartments of infected SH-SY5Y cells [Figure 4]a. This nuclear localization of ZIKV might be associated with loss of nuclear membrane in ZIKV-infected SH-SY5Y cells [Figure 4]a which was observd in 70% of the ZIKV-infected SH-SY5Y cell specimens (data not shown). In addition, the ZIKV particles observed within the cytoplasmic compartments of infected SH-SY5Y cells were visible like electron dense spherical viral particles (50-70 nm in diam), which represented a general feature of flavivirus particles [Figure 4]a,[Figure 4]b,[Figure 4]c. At 80,000*, a turquoise striped appearance was observed on the surface of the virus [Figure 4]d. Nevertheless, mitochondrial swelling was frequently observed with the presence of ZIKV particles in the cytoplasmic areas of infected SH-SY5Y cells [Figure 4]b.

Figure 4: Presence of Zika virus in cytoplasmic and nucleoplasmic compartments of infected SH-SY5Y cells, 12 h post-infection, indicated by intracellular spherical electron dense structure with 50–70 nm in size (). The presence of Zika virus particle in neucleoplasmic compartment may correlate to the loss of nuclear membrane of SH-SY5Y cell as indicated by { in (a); Mitochondrial swelling was frequently observed after infection as shown in (b) in the form of *; The size and shape (incomplete circular shapes) of the virus may depend on its stage as indicated in (c) through ↓. (d) Virus appearance at At 80,000×. The size of scale bars in (a) and (c) = 500 nm (White); in (b) = 1 μm; and in (d) = 50 nm (Black); N–Nucleus.

Zika virus has emerged as a severe health threat by virtue of its fast paced global spread and associated morbidities[24]. The growing incidence of ZIKV infection causing newborn microcephaly and adult neurological defects in humans (Guillain-Barré syndrome) during the recent years indicates that ZIKV is permissible to different types of human cells[3],[18],[25]. Zika virus was recently detected in the amniotic fluid of two pregnant women whose fetuses were diagnosed with microcephaly[26]. These studies suggest that ZIKV is a global public health concern for newborns as well as adults. However, little is known about direct cell targets and mechanisms of ZIKV infection especially in human adult neuronal cells.

In this study, human SH-SY5Y cell lines were used as an in vitro model to investigate the impact of ZIKV on human adult neuronal cells. It was observed that SH-SY5Y cells are permissive for ZIKV infection and replication. During the ZIKV infection, SH-SY5Y cell viability was rapidly reduced with increased cellular cytotoxicity. Moreover, the presence of high viral RNA copy number gradually increased during the initial period of infection, indicating active viral replication in the infected SH-SY5Y cells. The infection of ZIKV in SH-SY5Y cells is similar to that in skin fibroblasts[11]. Moreover, the observations of ZIKV infection were similar to the dengue infection in which DENV reduces host cell-viability by inducing the mechanism of apoptosis to facilitate the exocytosis of viral particles[27]. It can be suggested that ZIKV reduces SH-SY5Y cell viability by inducing apoptotic cell death like DENV. However, further study is required to elucidate the molecular mechanism of how ZIKV induces apoptotic cell death in adult human neuronal cells.

In general, type I interferon (IFN) provides a host antiviral innate immunity in vertebrates. However, flaviviruses (e.g. DENV, WNV, and YFV) induce several well-known mechanisms to antagonize the effectiveness of IFN and plays a critical role in spread of mosquito-borne diseases[28]. In the present study, SH-SY5Y and Vero cells infected with ZIKV were used to determine the cellular morphologies at different intervals post-infection. Earlier studies have used Vero cells for propagating viral particles in vitro as a standard procedure[29],[30]. But Vero cells are unable to synthesize interferon as its gene coding for interferon proteins is inactivated or deleted[31]. The results of this study showed that ZIKV induces more morphological changes in SH-SY5Y cells than Vero cells by using graded CPE method. Less morphological changes in Vero cells to ZIKV infection might be due to the lack of interferon system in Vero cells. A recent study has shown that ZIKV is permissive to human cells by directly targeting the human STAT2 to inhibit type I interferon signaling[20]. The study also suggests that unlike DENV, ZIKV does not require E3 ubiquitin ligase UBR4 to induce STAT2 degradation, indicating the remarkable function of ZIKV on IFN antagonism. Taken together, the present study demonstrated that SH-SY5Y cells provide several important host cell features during ZIKV infection including viral replication, host cell viability, cytotoxicity, and morphological characteristics. Thus, it could be suggested that SH-SY5Y cells can serve as an effective human adult neuronal cell-based model to further elucidate the ZIKV biology in infected adult human neurons.

During flavivirus infection, the virus enter into the host cells by using viral envelope protein that interacts with several host cell surface receptors. Earlier studies have shown that two distinct transmembrane receptors TIM and TAM proteins participate in the phagocytic engulfment and play an important role for internalizing DENV in human cells[32],[33]. Similar to DENV, our previous study has shown that ZIKV entry into human host cell is mediated by TIM-1 and TAM family member (AXL)[11]. Moreover, the electron microscopy analysis of ZIKV-infected primary human skin fibroblasts, showed that ZIKV viral capsid is found in an association with the endoplasmic reticulum, suggesting that ZIKV replication occurrs close to host cell membrane[11], while the replication of flaviviruses often takes place in the cytoplasm of the host[34].

In this study, electron microscope was used to determine the presence of ZIKV viral particle (as well its morphological features) in infected SH-SY5Y cells. Intracellular spherical electron dense structures with 50–70 nm in size as indicated for ZIKV was observed to be located in the endoplasmic reticulum and adjacent to the mitochondria of infected SH-SY5Y cells, similar to our earlier report of ZIKV-infected fibroblasts. Strikingly the study also revealed that ZIKV particle can be found within the nucleus of infected SH-SY5Y cells. The presence of ZIKV particle in the nucleoplasmic compartment of infected SH-SY5Y cells may correlate to the loss of host’s nuclear membrane (indicating that they might be involved in its destruction). A study has shown that Spondweni virus (SPOV), a mosquito-borne flavivirus closely related to ZIKV, is a flavivirus that infects the nucleus of the host cells. Moreover, SPOV non-structural protein (NS5-SPOV) has been found to inhibit the STAT2 mediated IFN-stimulated genes (ISGs) in the nucleus of the host cells during viral infection[20]. The nucleoplasmic ZIKV particle observed in this study might be using similar IFN antagonistic mechanism of SPOV to induce the neurological pathogenesis in human. However, the exact molecular mechanism used by ZIKV to infect adult neurons needs further research and investigation.

Conclusion

The study established an effective human adult neuronal-cell-based in vitro model to study the impact of Zika virus infection on adult human neurons. It demonstrated how ZIKV infected, replicated, and affected the SH-SY5Y cellular homeostasis. The presence of ZIKV particle inside the nucleus of infected SH-SY5Y cells, and the loss of nucleas membrane indicates that they might cross this membrane for multiplication by destroying it. Hence, SH-SY5Y cells could be used as an in vitro adult human neuronal cell-based model, for further elucidating the ZIKV biology, and highlight other possible significance of Zika virus distribution through nuclear localization, which may correlate to the neuropathological defects in ZIKV-infected adult humans. Although, the exact molecular mechanism by which ZIKV translocates into the nucleus of adult human neuronal hosts remains to be determined, the potential role of how ZIKV induces adult human neuronal defects via nuclear translocation is clearly highlighted through this study. Further, research is necessary to elucidate how ZIKV induces other cellular responses on adult human neuronal cells for developing an effective therapeutic intervention in the near future.

Conflict of interest

The authors declare that they do not have any conflict of interest.

Acknowledgements

The authors are thankful to the AFRIMS and the Ministry of Public Health, Thailand for providing ZIKV isolates. The research chair grant from the National Science and Technology Development Agency, and the scholarship from Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. program (Grant No. PHD/0001/2557) to N.L. and S.S. are gratefully acknowledged.